Reaction of M1G With Hydrazines
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 317
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bases. Exhaustive efforts to eliminate this artifact in-
cluding purification of PFBH as well as the use of
ultrapure organic solvents and water were unsuccessful.
The origin of the low levels of pyrazole is not clear, but
it is important to note that PFBP is formed in the
complete absence of DNA constituents in the reaction
with PFBH. Despite this complication, the sensitivity
obtainable with the procedure described is adequate for
most routine applications. Given that the method is
suitable for automated operation, the assay may be useful
for analysis of a broad range of DNA samples. Indeed,
this single analytical method can be used for the analysis
of nucleic acid and protein conjugates.
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We demonstrated the utility of the PFBH assay by
reacting bleomycin with DNA then analyzing for M1G/
oxopropenyl/MDA equivalents by reaction with PFBH.
The DNA was not purified or dialyzed after reaction with
bleomyin to increase the probability of detecting reactive
equivalents present on DNA or released following strand
scission (e.g., base propenals). A direct relation was
observed between bleomycin concentration and the extent
of generation of MDA equivalents (MDA, base propenals,
M1G, M1A, etc.). At the highest concentration of bleomy-
cin used (200 µM), the level of MDA/oxopropenyl equiva-
lents was 1.25/102 bases or approximately 1 equiv/100
bases. Parallel analyses of these samples for M1G by
immunoslotblot indicated that the content of this adduct
represented only 0.008% of the total MDA/oxopropenyl
equivalents generated. Thus, most of the reactive moi-
eties detected by the PFBH assay must be base pro-
penals, MDA, or related molecules that are unable to
form additional M1G. This inefficiency of M1G production
from in situ generated MDA or base propenals may
reflect the low reactivity of the conjugate base of MDA
at neutral pH or conformational restrictions on the
reaction of base propenals with DNA.
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of the pyrimidopurinone adduct of deoxyguanosine by base
propenal. Proc. Natl. Acad. Sci. U.S.A. 95, 11113-11116.
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A. P. (1981) Bleomycin-induced strand-scission of DNA. Mecha-
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involved in the formation of sequence-dependent bistranded DNA
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(2000) Reactivity and mutagenicity of endogenous DNA oxo-
propenylating agents: base propenals, malondialdehyde, and
N(epsilon)-oxopropenyllysine. Chem. Res. Toxicol. 13, 1235-1242.
(18) Niedernhofer, L. J ., Riley, M., Schnetz-Boutaud, N., Sanduwaran,
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Temperature-dependent formation of a conjugate between tris-
(hydroxymethyl)aminomethane buffer and the malondialdehyde-
DNA adduct pyrimidopurinone. Chem. Res. Toxicol. 10, 556-561.
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oxynucleotide containing the alkaline labile malondialdehyde-
deoxyguanosine adduct pyrimido[1,2-R]purin-10(3H)-one. J . Am.
Chem. Soc. 117, 5007-5008.
The present report expands the repertoire of reactions
of M1G in DNA toward nucleophiles and utilizes the
findings to provide a robust and straightforward assay
for the presence of oxopropenyl equivalents in DNA. Since
a number of drugs generate free radicals capable of
degrading DNA to products with oxopropenyl equiva-
lents, this assay should find broad use as a convenient
method for quantifying oxidative DNA damage.
(20) Schnetz-Boutaud, N., Daniels, J . S., Hashim, M. F., Scholl, P.,
Burrus, T., and Marnett, L. J . (2000) Pyrimido[1,2-R]purin-10-
(3H)-one: A reactive electrophile in the genome. Chem. Res.
Toxicol. 13, 967-970.
(21) Mao, H., Schnetz-Boutaud, N. C., Weisenseel, J . P., Marnett, L.
J ., and Stone, M. P. (1999) Duplex DNA catalyzes the chemical
rearrangement of a malondialdehyde deoxyguanosine adduct.
Proc. Natl. Acad. Sci. U.S.A. 96, 6615-6620.
(22) Mao, H., Reddy, G. R., Marnett, L. J ., and Stone, M. P. (1999)
Solution structure of an oligodeoxynucleotide containing the
malondialdehyde deoxyguanosine adduct N2-(3-oxo-1-propenyl)-
dG (ring-opened M1G) positioned in a (CpG)3 frameshift hotspot
of the Salmonella typhimurium hisD3052 gene. Biochemistry 38,
13491-13501.
Ack n ow led gm en t. This work was supported by a
research grant (CA77839 and CA87819) and center grant
from the National Institutes of Health (ES00267). M.
Otteneder was the recipient of a postdoctoral fellowship
from the Deutscher Akademischer Austauschdienst.
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